CN104818220A - Rhizopus oryzae strain JHSW01 obtained by screening rotten straws - Google Patents
Rhizopus oryzae strain JHSW01 obtained by screening rotten straws Download PDFInfo
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Abstract
The invention provides a rhizopus oryzae strain JHSW01 obtained by screening rotten straws, and the preservation number of the strain is CCTCCM 2015189. The strain has the capacity of generating high live saccharifying enzyme and generating cellulose, and is capable of effectively promoting the synthesis and secretion of the flora cellulose in the process of promoting forestry and agricultural residues to rot; the rhizopus oryzae strain JHSW01 obtained by screening the rotten straws has the characteristics of being capable of effectively decomposing cellulose, fully using renewable resources such as straws by using the rhizopus oryzae strain JHSW01 to ferment the straws, and solving the pollution problem on environment caused by direct incinerating the straws; furthermore, a media component adopted by the rhizopus oryzae strain JHSW01 is simple and liable to prepare, thereby being easy to prepare and beneficial for cost saving of industrial production; in addition, the culture condition of the train is easy to control and convenient to industrially apply, therefore the good market application prospect can be realized.
Description
Technical field
The invention belongs to biological technical field, be specifically related to a strain from rotten stalk, screen the Rhizopus oryzae bacterial strain JHSW01 obtained.
Background technology
Agricultural crop straw is first renewable resources on the earth, and China's owning amount occupies first place in the world.In annual about more than the 600000000 tons of agricultural crop straws produced in the whole nation, by mechanize, also field and stack retting decomposition into field utilize agricultural crop straw about 200,000,000 tons, adopt ensiling, ammonification waited abdomen also field mode utilize about 200,000,000 tons, a small amount of agricultural crop straw is also had to be used for gasification and processing etc. in addition, 2/3 of stalk total amount achieves comprehensive utilization substantially, also have an appointment 1/3, namely about 200,000,000 tons residue stalks are not yet fully utilized, some peasants take the simplest processing mode-burning, cause topsoil, soil mineralization, the social economy that fire failure etc. are a large amount of and ecological problem, cause the extensive concern of the whole society.
Stalk itself mainly contains Mierocrystalline cellulose, hemicellulose and xylogen three kinds of material compositions, also containing a small amount of protein and mineral element, is a kind of good microorganism growth substrate.And Rhizopus oryzae is one of important mould in middle traditional Chinese medicines and distiller's yeast, a large amount of amylase, saccharifying enzyme, proteolytic enzyme can be produced, Mierocrystalline cellulose in decomposing straw and hemicellulose, be translated into the organic acids such as lactic acid, existing large quantities of scholar starts to utilize stalk cooking liquor to carry out the experiment of Rhizopus oryzae lactic acid-fermenting, and the sugar of 80% can be converted into lactic acid by this bacterial classification under suitable aerobic conditions to have article to point out.But this bacterial classification is less for the application of the fermentation sofening treatment of agriculture and forestry organic waste material at present.
Summary of the invention
The object of this invention is to provide a strain from rotten stalk, screen the Rhizopus oryzae bacterial strain JHSW01 obtained, the Rhizopus oryzae bacterial strain JHSW01 that the present invention screens produces food fermented bacterium used as a kind of, not only there is no toxic side effect, and Rhizopus oryzae fermentation is fast, thalli growth nutritional requirement is simple, may be used for the agriculture and forestry organic waste material that ferments, there is good market application foreground.
For achieving the above object, the present invention adopts following technical proposals to be achieved:
The Rhizopus oryzae bacterial strain JHSW01 obtained is screened in one strain from rotten stalk, and its Classification And Nomenclature is
rhizopus oryzae, its deposit number is CCTCC M 2015189.
Described Rhizopus oryzae bacterial strain JHSW01 Gram-positive; Bacterium colony is dense, and mycelia crawls to creep, initial in white, after become chocolate; Rhizoid is flourishing, and branch is root shape, in chocolate; Fibrillae of spores has Chlamydospores, sporangiophore 2 ~ 4 bunchy, sporangiophore uprightly has branch, afterwards in black.
Further, described Rhizopus oryzae bacterial strain JHSW01 is inoculated in liquid nutrient medium to cultivate and produces the saccharifying enzyme of enzyme work up to 110-200 U/mL.
Further, described Rhizopus oryzae bacterial strain JHSW01 is inoculated in solid medium to cultivate and produces the cellulase that enzyme work reaches 50 ~ 100U/g.
Further, described Rhizopus oryzae bacterial strain JHSW01 has the characteristic of effective decomposition of cellulose and hemicellulose.
Compared with prior art, advantage of the present invention and positively effect are: the present invention screens and obtains a Rhizopus oryzae bacterial strain JHSW01 from rotten stalk, through experimental verification its can cellulase-producing and there is the ability producing high saccharifying enzyme and live, can effectively decomposition of cellulose and hemicellulose; Utilize the Rhizopus oryzae bacterial strain JHSW01 that screens can effective cellulolytic characteristic, the agriculture and forestry organic waste materials such as fermented stalk can be used it for, not only take full advantage of this renewable resources of stalk, and solve stalk and directly burn the pollution problem that environment is brought; The culture medium prescription that Rhizopus oryzae bacterial strain JHSW01 of the present invention adopts is simple and easy is easy to preparation, is conducive to industrial cost savings; Spawn culture condition of the present invention is simply easy to control, and is convenient to industrial applications, so have good market application foreground.
After the specific embodiment of the present invention, the other features and advantages of the invention will become clearly.
Accompanying drawing explanation
Fig. 1 is the slat chain conveyor figure of Rhizopus oryzae JHSW01 in the present invention.
Embodiment
Below in conjunction with the drawings and specific embodiments, technical scheme of the present invention is described in further detail.
Embodiment 1
One, the separation screening of Rhizopus oryzae bacterial strain JHSW01
On July 4th, 2010 samples more than 50 parts from Yinan, Shandong great Zhuan wheat rots stalk, gets 6g sample powder respectively and is immersed in 50 mL physiological saline, and under 20 DEG C of conditions, every 2h concussion once.Get 100 μ L sample suspension respectively at nutrient agar plate even spread, 3 ~ 7d is cultivated in 28 DEG C of constant incubators, the typical single bacterium colony method of scoring of picking form is inoculated on nutrient agar plate, obtains pure bacterium, filter out 8 strain moulds altogether by repeated multiple times line.
8 obtained strain moulds are inoculated in liquid PDA substratum with transfering loop and cultivate.The wherein strain bacterium called after Rhizopus oryzae JHSW01 obtained, it meets the biological characteristics of Rhizopus oryzae, is specially: Gram-positive; Bacterium colony is dense, and mycelia crawls to creep, initial in white, after become chocolate; Rhizoid is flourishing, and branch is root shape, in chocolate; Fibrillae of spores has Chlamydospores, sporangiophore 2 ~ 4 bunchy, sporangiophore uprightly has branch, afterwards in black, see accompanying drawing 1.
Two, the bacteria molecule genetic classification qualification of Rhizopus oryzae bacterial strain JHSW01
First extract the DNA of bacterial strain JHSW01, with this DNA for template, 18S rDNA universal primer is primer, increases to 18S rDNA fragment.The fragment of amplification is directly carried out sequencing.Compared by blast program on the result of order-checking input NCBI website, the 18s rDNA sequence homology degree that result shows the Rhizopus oryzae in this bacterial strain 18S rDNA sequence and GenBank gene pool in Rhizopus is the highest, and homology reaches 100%.Phylogenetic analysis is carried out by the 18S rDNA sequence of DNAMAN6.0 to bacillus existing in Genbank, result shows, 18S rDNA and the Rhizopus oryzae homology of bacterial strain JHSW01 are the highest, therefore can judge the Rhizopus oryzae of this bacterial strain as Rhizopus.
Through described Identification of Biological Characteristics and the qualification of bacteria molecule genetic classification authentication method, can determine that screened bacterial strain JHSW01 is Rhizopus, Rhizopus oryzae.The Rhizopus oryzae bacterial strain JHSW01 screened is carried out culture presevation, depositary institution: China typical culture collection center (CCTCC); Address: Luojiashan, Wuchang, Wuhan City, Hubei Province, Wuhan University's preservation center; Preservation date: on April 2nd, 2015, Rhizopus oryzae (
rhizopus oryzae) deposit number be CCTCC M 2015189.
Three, Rhizopus oryzae bacterial strain JHSW01 produces the work of high saccharifying enzyme during the fermentation
Take wheat bran 2.4g, soy peptone 1.6g, Zulkovsky starch 0.8g, K
2hPO
40.16g, MgSO
4.7H
2after O 0.04g, KCl 0.04g is dissolved in the distilled water of 80mL, be placed in 250mL triangular flask, 121 DEG C of sterilizing 20min.After cooling, inoculation two ring inclined-plane Rhizopus oryzae JHSW01 mycelium, are placed in 30 DEG C, 190rpm condition bottom fermentation 40h, and centrifuging and taking 1mL supernatant liquor measures enzyme and lives.The qualification result utilizing DNS method to carry out Rhizopus oryzae glucoamylase enzyme alive is as follows.
The glucoamylase enzyme experimental result alive of table 1 Rhizopus oryzae JHSW01
| Numbering | 1 | 2 | 3 | Average enzyme is lived |
| Rhizopus oryzae group | 112.21U/g | 112.83 U/g | 113.06 U/g | 112.7U/g |
As can be seen from Table 1, this Rhizopus oryzae can produce higher saccharifying enzyme and lives being inoculated in substratum fermentation, through test, its saccharifying enzyme work proves that the saccharifying enzyme that can reach 110-200 U/mL is lived.
Four, the mensuration that Rhizopus oryzae bacterial strain JHSW01 fermented stalk cellulase-producing is alive
Rhizopus oryzae enzymatic productivity is measured according to carboxymethyl cellulose (reducing sugar method) enzyme activity (CMCA-DNS) measuring method in " People's Republic of China's light industry industry standard " QB2583-2003.
Experimental program is: get 3g stalk powder and 0.1g urea joins in the 500mL triangular flask containing 100mL distilled water, 121 DEG C of sterilizings, 20min.Cooling access 2 ring Rhizopus oryzae JHSW01 mycelium, what bacterium control group does not take over.Fermentation system, at 30 DEG C, cultivates 96h under 190rpm condition, gets the cellulase activity that fermented supernatant fluid measures two groups of fermentation systems.Experiment is provided with 4 groups of experiments altogether, and wherein three groups is the parallel laboratory test of inoculation Rhizopus oryzae, and another group is the control group not connecing bacterium, and experimental result is as shown in table 2:
The cellulase activity experimental result of table 2 Rhizopus oryzae JHSW01
| Numbering | 1 | 2 | 3 | Average enzyme is lived |
| Rhizopus oryzae group | 58.31U/g | 59.87 U/g | 59.46 U/g | 59.21U/g |
Control group inside after sterilizing does not have bacterial growth, therefore do not produce cellulose enzyme activity, but the reducing sugar amount that control group produces after can providing straw under high temperature sterilizing, so all removed this reducing sugar impact produced in sterilization process of stalk powder before experiment enzyme activity determination, cellulase can be produced in Rhizopus oryzae fermenting process as can be seen from Table 2.
Five, the promoter action that in mixed fermentation, Rhizopus oryzae JHSW01 produces cellulase
Take the 100g stalk powder two parts of water ratio 58.3%, add 5g urea and 200g water respectively, inoculum size is for such as joining 15mL bacterium liquid in 100g stalk powder than 15%(volume mass according to volume mass), be stacked in the beaker of 2L, be placed in the thermostat water bath 90h of 30 DEG C, in fermenting process, every 24h turning once.Fermentation arrange altogether one group of experiment and one group of contrast (control group be the Rhizopus oryzae seed liquor of 15% is replaced with 15% distilled water), the pH4.8 that 1g stalk powder adds 9mL is got after fermentation ends, the citric acid-sodium citrate damping fluid of 0.05mol/L, soak 30min, centrifuging and taking supernatant liquor measures cellulase activity.
Rhizopus oryzae enzymatic productivity is measured according to carboxymethyl cellulose (reducing sugar method) enzyme activity (CMCA-DNS) measuring method in " People's Republic of China's light industry industry standard " QB2583-2003.Experimental result is as shown in table 3:
Table 3: the promoter action experimental result that in mixed fermentation, Rhizopus oryzae produces cellulase
| 1 | 2 | 3 | Average enzyme is lived | |
| Control group | 85.62U/g | 86.01U/g | 85.98U/g | 85.87U/g |
| Rhizopus oryzae group | 142.31U/g | 140.87 U/g | 143.06 U/g | 142.08U/g |
As can be seen from Table 3, the cellulase activity added in Rhizopus oryzae secondary fermentation stalk reaches 142.08U/g, 1.65 times have been exceeded than the fermentation system not adding Rhizopus oryzae, because this Rhizopus oryzae self cellulase-producing enzyme amount is lower, can find out that interpolation Rhizopus oryzae has positive influence for the secretion of stalk fermentation flora cellulase-producing thus, this is this Rhizopus oryzae sugar that can effectively utilize fermentative microflora decomposition of cellulose and hemicellulose to produce mainly, be translated into lactic acid, reduce substrate inhibition, be conducive to the secretion promoting fermentative microflora cellulase.
Above embodiment only in order to technical scheme of the present invention to be described, but not is limited; Although with reference to previous embodiment to invention has been detailed description, for the person of ordinary skill of the art, still can modify to the technical scheme described in previous embodiment, or equivalent replacement is carried out to wherein portion of techniques feature; And these amendments or replacement, do not make the essence of appropriate technical solution depart from the spirit and scope of the present invention's technical scheme required for protection.
Claims (5)
1. the Rhizopus oryzae bacterial strain JHSW01 obtained is screened in a strain from rotten stalk, it is characterized in that: its Classification And Nomenclature is
rhizopus oryzae, its deposit number is CCTCC M 2015189.
2. Rhizopus oryzae bacterial strain JHSW01 according to claim 1, is characterized in that: described Rhizopus oryzae bacterial strain JHSW01 Gram-positive; Bacterium colony is dense, and mycelia crawls to creep, initial in white, after become chocolate; Rhizoid is flourishing, and branch is root shape, in chocolate; Fibrillae of spores has Chlamydospores, sporangiophore 2 ~ 4 bunchy, sporangiophore uprightly has branch, afterwards in black.
3. Rhizopus oryzae bacterial strain JHSW01 according to claim 1, is characterized in that: described Rhizopus oryzae bacterial strain JHSW01 is inoculated in liquid nutrient medium to cultivate and produces the saccharifying enzyme of enzyme work up to 110-200 U/mL.
4. Rhizopus oryzae bacterial strain JHSW01 according to claim 1, is characterized in that: described Rhizopus oryzae bacterial strain JHSW01 is inoculated in solid medium to cultivate and produces the cellulase that enzyme work reaches 50 ~ 100U/g.
5. Rhizopus oryzae bacterial strain JHSW01 according to claim 1, is characterized in that: described Rhizopus oryzae bacterial strain JHSW01 has the characteristic of effective decomposition of cellulose and hemicellulose.
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Cited By (2)
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| CN110972595A (en) * | 2019-12-11 | 2020-04-10 | 扬州大学 | Method for returning rice straws to field in cold region and autumn |
| CN111334440A (en) * | 2020-04-16 | 2020-06-26 | 浙江树人学院（浙江树人大学） | Rhizopus oryzae CH5 and application thereof in extraction of ganoderma sinensis polysaccharide |
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| CN111334440A (en) * | 2020-04-16 | 2020-06-26 | 浙江树人学院（浙江树人大学） | Rhizopus oryzae CH5 and application thereof in extraction of ganoderma sinensis polysaccharide |
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